X-ray crystallography based experiments of this proposal are aimed to understand the interaction of Par6C with GTPases Rit and Rin, and the role of CDC42 in modulating this event. GTPases Rit and Rin are unique members of the Ras subfamily. They possess a divergent G2 effector binding motif, lack the prenylation motif, and can undergo rapid intrinsic nucleotide (GDP?GTP) exchange. Both GDP and GTP bound states of Rit and Rin can bind to Par6C, a cell polarity regulator. The ability of high nucleotide exchange by these GTPases may be the causative factor that allows Par6C to bind both states. Hence it is important to compare the molecular architectures of the active (GTP) and inactive (GDP) states of these GTPases using X-ray crystallography (Aim 1). Par6C has a PDZ domain through which Par6C interacts with Rit and Rin, and therefore the structures of PDZ:RitGDP and PDZ:RitGppNHp complexes will be solved (Aim 2). These novel structures will identify the unknown mode by which a PDZ domain recognizes GTPases. Experimental evidence indicates that the isolated PDZ domain of Par6C binds to both nucleotide states of Rit and Rin with equal affinity but full length Par6C has a slight higher preference to the GTP bound state. This indicates other molecular segments in the full length Par6C can modulate the PDZ:GTPase interaction. This will be investigated solving the structure of Par6C:RitGppNHp complex and comparing it with the structure of the isolated PDZ domain bound to Rit (Aim 2). In addition to the PDZ domain Par6 has a semi-CRIB motif through which CDC42, a Rho GTPase, interacts and induces conformational changes in the adjacent PDZ domain. CDC42 bound full length Par6C can only bind to the GTP bound state of Rit or Rin. To study the role of CDC42 in regulating the interaction of Par6C with Ras GTPases we will determine, through biochemical experiments how CDC42 regulates Par6C:Rit complexes and identify crystallization conditions for the RitGppNHp:Par6C:CDC42GppNHp complex (Aim 3). Lastly our proposal attempts to modulate the nucleotide exchange on Rit GTPases using drug compounds. We will determine the mechanism by which ALS-576 a small organic molecule inhibits nucleotide exchange on Rit and its interaction with Par6C. We would identify conditions that crystallize the complex ALS-576: Rit and crystallize the same (Aim 4). PUBLIC HEALTH RELEVANCE: The project attempts to understand the role of Rho GTPase in controlling the interaction between Ras GTPase and a cell polarization protein Par6C using X-ray crystallography, biochemical and other biophysical methods.